1. Field of the Invention
This invention relates to a novel live attenuated FMD vaccine comprising mutated virulence determinants within the coding region of the SAP domain of the leader protein of FMDV wherein the mutated virulence determinants of the novel live FMD vaccine confer significant attenuation in swine and cattle as evidenced by absence of clinical disease, the induction of a significant FMDV-specific neutralizing antibody response, and protection against subsequent homologous virus challenge; and to the method of treating swine, cattle, goats, and sheep with the vaccine in order to protect swine, cattle, goats, and sheep against clinical FMD.
2. Description of the Relevant Art
Foot-and-mouth disease virus (FMDV) is the etiologic agent of FMD, a highly contagious disease that affects wild and domestic cloven-hoofed animals, including swine, cattle, sheep, goats, deer, and buffalo that quickly replicates in the host spreading to susceptible animals by contact and aerosol (Grubman and Baxt. 2004. Clinical Micro. Rev. 17: 465-493). FMD is considered one of the most contagious diseases of animal or man. The International Organization of Animal Health (OIE) includes FMD in the list A of diseases which requires the immediate official report of confirmed FMD cases and cessation of trading of susceptible animals including their products. FMD is enzootic in all world continents except for Australia and North America. Although the U.S. has been FMD-free since 1929 recent natural outbreaks in previously disease-free countries and the rapid advancement of world globalization, have significantly increased public awareness about this disease. Outbreaks in Taiwan (1997) and in the UK (2001, 2007) have resulted in losses to the agricultural industry surpassing $15 billion with more than 10 million animals slaughtered. A chemically inactivated vaccine is currently used in enzootic areas (Doel, T. R. 2003. Virus Res. 91:81-99) but FMD-free countries are reluctant to use this vaccine for several reasons: vaccine manufacturing requires a biosafety level 3 (BSL3) containment facility, the vaccine does not allow for differentiation between vaccinated and infected animals and there is a potential risk of deriving asymptomatic disease carriers upon exposure of vaccinated animals to infectious virus. As a result the OIE requires that countries that vaccinate to control FMD must wait 6 months after demonstrating, by serosurveillance, the absence of FMD before regaining FMD-free status, while countries that slaughter or vaccinate and then slaughter must only wait 3 months. Mayr et al. (2001. Vaccine 19:2152-2162) and Moraes et al. (2002. Vaccine 20:1631-1639) have constructed a recombinant vaccine delivered by a replication defective human adenovirus type 5 (Ad5) vector that can protect cattle and swine from clinical disease and viremia, allows unequivocal differentiation of vaccinated from infected animals, and does not require high containment facilities for vaccine production. However, similar to the inactivated vaccine, the Ad5 vaccine requires at least one week to induce a protective immune response and currently it is quite expensive to manufacture.
It has been reported that rapid and long lasting protection against viral infection is usually best achieved by vaccination with live-attenuated viral vaccines. Unfortunately, attempts to develop live-attenuated FMD vaccines have met with limited success. Instability of the mutant phenotype, excessive attenuation that results in failure to induce a protective immune response, differences in the degree of attenuation for individual species (e.g., swine vs. cattle) and the possibility of reversion to virulence, have stalled efforts in pursuing such an approach (Mowat et al. 1962. Nature 196:655-656; Mowat et al. 1969. Arch. Virol. 26:341-354; Martin and Edwards. 1965. Res. Vet. Sci. 36:196-201; Zhidkow and Sergeev. 1969. Veterinariia 10:29-31). With the advent of infectious FMDV cDNA it has been possible to introduce specific changes in the FMDV genome and evaluate the phenotypic changes after growth in cell culture and animals. As a result it is possible to test such virus variants as potential live-attenuated vaccine candidates (Rieder et al. 1993. J. Virol. 67:5139-5145; Rieder et al. 1994. J. Virol. 68:7092-7098; Piccone et al. 1995a. J. Virol. 69:5376-5382).
The virus is the prototype member of the Aphthovirus genus of the Picornaviridae family and consists of a positive strand RNA genome of about 8 kb surrounded by an icosahedral capsid containing 60 copies each of four structural proteins. Upon infection, the viral RNA is translated as a single polyprotein which is concurrently processed by three viral-encoded proteinases, leader (Lpro), 2A and 3Cpro into precursors and mature structural (VP1, VP2, VP3 and VP4) and non structural proteins (Lpro), 2A, 2B, 2C, 3A, 3B, 3Cpro and 3Dpol) (Rueckert, R. R. 1996. In: Field's Virology, Fields et al. (eds), Lippincott-Raven, Philadelphia and New York, pages 609-654).
Studies in our laboratory have demonstrated that Lpro plays a critical role in the pathogenesis of FMDV. In a hallmark discovery it was shown that deletion of the portion of the viral genome coding for the L protein region results in a viable attenuated, not transmissible (leaderless) virus that induced partial protection against challenge (Mason et al. 1997. Virology 227:96-102; Chinsangaram et al. 1998. Vaccine 16:1516-1522). Studies with this virus have significantly contributed to understanding some of the molecular mechanisms involved in FMDV virulence. The FMDV L protein is positioned at the N-terminus of the viral polyprotein. Translation of the polyprotein is initiated at two different AUGs which are separated by 84 nucleotides yielding two alternative forms of Lpro). Initiation at the first AUG results in Lab, an Lpro form of 201 amino acids, and initiation at the second AUG results in Lb, an Lpro form of 173 amino acids which is predominantly produced (Cao et al. 1995. J. Virol. 69:560-563; Piccone et al. 1995b. J. Virol. 69:4950-4956).
The FMDV L protein is a protease that in addition to cleaving itself from the nascent viral polyprotein, cleaves cellular proteins and modulates the host innate immune response (Strebel and Beck. 1986. J. Virol. 58:893-899; Devaney et al. 1988. J. Virol. 62: 4407-4409; Chinsangaram et al. 1999. J. Virol. 73: 9891-9898; Chinsangaram et al. 2001. J. Virol. 75: 5498-5503; de los Santos et al. 2006. J. Virol. 80:1906-1914; de los Santos et al. 2007. J. Virol. 81:12803-128151).
One of the reasons for the attenuation of the leaderless virus is the inability of this virus to block host cell translation, in particular, translation of type I interferon (IFNα/β) (Chinsangaram et al. 1999, supra). In most cell types, expression of IFN is induced in response to viral infection. Subsequently, IFN protein is secreted and binds to specific cell-surface receptors acting in an autocrine or paracrine manner. The interaction between IFN and its receptor induces a series of signal transduction events that lead to the expression of interferon stimulated genes (ISGs) which have antiviral and/or antiproliferative properties (Haller et al. 2006. Virology 344:119-130; Honda et al. 2006. Int. Immunol. 17:1367-1378). Among the ISGs, the IFN induced dsRNA dependent protein kinase (PKR) and the IFN induced ribonuclease L (RNase L) have been shown to inhibit FMDV replication (Chinsangaram et al. 2001, supra; de los Santos et al. 2006, supra). Therefore, the Lpro inhibition of host translation limits the synthesis of IFN protein and the IFN-triggered antiviral effects.
Recent data has demonstrated that Lpro, in addition to its effect on translation, also blocks the induction of IFNβ transcription, a very early response to viral infection (de los Santos et al. 2006, supra). In uninfected cells, transcription of IFNβ is not detectable, but upon viral infection latent transcription factors, including nuclear factor KB (NF-κB), interferon regulatory factors 3 and 7 (IRF3, IRF7) and the activating transcription factor 2/cellular Jun protein complex (ATF2/cJun, also named AP-1) are activated and translocated from the cytoplasm to the nucleus of the cell, where they bind to their respective IFNβ enhancer elements, thereby inducing gene expression (Honda et al., supra). Several studies have shown that one of the mechanisms employed by different viruses to antagonize the innate immune response is the inhibition of the induction of IFNβ transcription (Conzelmann, K.-K. 2005. J. Virol. 79: 5241-5248; Haller et al., supra). Among picornaviruses, it has been reported that poliovirus causes the degradation of several proteins, including the p65/RelA subunit of NF-κB and the RNA helicase MDA-5, resulting in reduced IFNβ transcription (Barral et al. 2007. J. Virol. 81:3677-3684; Neznanov et al. 2005. J. Biol. Chem. 280:24153-24158).
Our group has shown that during FMDV infection down-regulation of IFNβ transcription is associated with Lpro dependent degradation of the p65/RelA subunit of NF-κB (de los Santos et al. 2006, supra; de los Santos et al. 2007, supra). Interestingly, our studies showed that Lpro translocates to the nucleus of infected cells and there is a correlation between the translocation of Lpro and the decrease in the amount of nuclear p65/RelA. However, it still remains unclear how FMDV Lpro induces p65/RelA degradation since highly conserved Lpro cleavage sites have not been found in the p65/RelA protein primary sequence nor have defined p65/RelA degradation products been detected during FMDV infection (de los Santos et al. 2007, supra).
Recently, the availability of bioinformatic tools has resulted in the prediction of multiple domains within the L protein, one of which is a putative SAF-A/B, Acinus and PIAS (SAP) domain, between amino acids 47 and 83, (following the numbering from the Lb form of Lpro). This domain, SAP, has been described in other proteins which are involved in transcriptional control (Aravind and Koonin. 2000. TIBS 25:112-114).
Here we report the effects of a double point mutation in the coding region of the functional SAP domain of FMDV Lpro and complete attenuation in vivo. Animals infected with the doubly mutated virus were protected when challenged with virulent FMDV. Such attenuated viruses permit the rational design of live attenuated FMD vaccines. Live-attenuated FMD vaccines can potentially induce longer protection than current vaccines thereby reducing the need for bi- or tri-annual vaccination to ensure protection. Thus, there is a need for new FMD vaccines that display a stable and a significant attenuated phenotype which can be used to protect domestic cloven-hoofed animals (e.g., swine, cattle, goats, and sheep) from FMD.